1. Kinetic Folding Mechanism of Erythropoietin
Douglas D. Banks, Joanna L. Scavezze, Christine C. Siska 
Biophysical Journal 
Volume 96, Issue 10, Pages (May 2009)
DOI: /j.bpj
Copyright © 2009 Biophysical Society Terms and Conditions

2. Figure 1
Ribbon diagram of EPO with helices lettered A–D from the N-terminus. Disulfide bonds are shown in yellow, and tryptophan residues are shown in dark blue. Trp-88 is located on the short loop connecting the antiparallel second and third helices (BC loop) and has been reported to be largely quenched by the disulfide bond bridging cysteines 29 and 33 within the N-terminal portion of the first crossover loop (27). The figure is derived from the structure of a nonglycosylated EPO mutant (1BUY.pdb) described elsewhere (4) and rendered using DSviewerPro 6.0.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2009 Biophysical Society Terms and Conditions

3. Figure 2
(A) Representative equilibrium unfolding of EPO monitored by far-UV CD at 222 nm (open circles) and intrinsic tryptophan FL at 325 nm (solid triangles) were best fit locally to two-state unfolding model (solid lines). (B) These two data sets, along with an additional CD unfolding data set and a FL refolding data set (open boxes and triangles, respectively), were fit globally to the same model and are shown normalized to the fraction unfolded. The solid line represents the global fit and the fitted parameters are given in Table 1. The residuals between the data and the fits are shown in the panels below. Conditions are described in the legend to Table 1.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2009 Biophysical Society Terms and Conditions

4. Figure 3
Representative stopped-flow and manual mixed EPO folding and unfolding kinetic traces monitored by far-UV CD at 222 nm and intrinsic tryptophan FL. (A) SF-FL data monitored with a 320 nm cutoff filter was best fit to a sum of three exponential equations. The residuals of this fit and of a single and double exponential fit are overlaid for comparison in the panel below as black, dark gray, and light gray lines, respectively. The inset is a magnification of the initial time points of the folding reaction. (B) The kinetic amplitudes of SF-FL folding. The solid circles, squares, and triangles represent folding from the fast, intermediate, and slow phases respectively. (C) Manual mixing folding monitored by FL at 325 nm was best fit to a single exponential equation and showed evidence of a hyper-fluorescent intermediate populated within the time of mixing. The numbers corresponding to each trace indicate the final urea concentration EPO was folded to. (D) Unfolding of EPO to 5.8 M urea by manual mixing monitored by CD. The solid line represents the local fit of the data to a single exponential equation with residuals of the fit shown in the inset. The horizontal line is the folded signal at the same EPO concentration and an extrapolation of this signal to the end of the unfolding time. Conditions are described in the legend to Table 1.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2009 Biophysical Society Terms and Conditions

5. Figure 4
(A) Urea concentration dependence of the folding and unfolding reactions. Local fits of the SF-FL folding data are represented by solid squares (inset), solid circles, and open triangles. All manual mixing FL and CD data is shown by open circles and squares, respectively. Error bars represent the standard deviation of multiple kinetic traces and are smaller than all of the manual mixing data points. (B) SF-FL burst-phase analysis. The solid triangles are the FL signals calculated at the time of mixing from kinetic refolding traces that have been normalized to the folded baseline region of an equilibrium unfolding curve generated using a 320 nm cutoff filter (open boxes). This curve was fit to a two-state equilibrium model (solid line) to better define the unfolded baseline region (dashed line). Error bars represent the standard deviations of three refolding experiments and three equilibrium curves. Conditions are described in the legend to Table 1.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2009 Biophysical Society Terms and Conditions

6. Figure 5
(A) Three possible three-state folding mechanisms that describe a two exponential process. The intermediate (I) may be either on-pathway, off-pathway, or folding may proceed directly to the native state (N) from the unfolded ensemble (U), as well as through an intermediate in a parallel pathway mechanism. The microscopic rate constants are shown above and below the arrows. (B) Urea concentration dependence of the folding and unfolding reactions fit to a three-state on-pathway model. The microscopic rate constants (solid black lines) were calculated using Eq. 4 and used as initial parameters for fitting the two observed rate constants (red dashed and solid lines) using the on-pathway mechanism, Eq. 5. All data points are the same as those described in Fig. 4A. Fitted values of the rate constants and m‡-values are given in Table 1. Conditions are described in the legend to Table 1.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2009 Biophysical Society Terms and Conditions

7. Figure 6
Urea dependence of the two observable rate constants fit to an off-pathway model (A) and to a parallel or triangle mechanism (B). Fits of the two observable rate constants are shown as red dashed and solid lines. All data points are the same as those described in Fig. 4A.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2009 Biophysical Society Terms and Conditions

8. Figure 7
Free energy diagram of EPO folding and unfolding at 0 M, 3 M, and 6 M urea concentrations. Energy levels were calculated using the parameters in Table 1 and Eqs. 6 and 7. The reaction coordinate describes the amount of burial of surface area relative to the native state or βT value calculated using Eq. 8.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2009 Biophysical Society Terms and Conditions